The invention relates to a process for obtaining acyloins by enzymatic conversion of .alpha.-ketocarbon acid and/or aldehydes in the presence of pyruvate-decarboxylase (PDC) and it comprises a PDC suitable therefor as well as their production and the gene coding them.
Acyloins, or respectively, x-hydroxy ketones are compounds with an optically active C-atom, which play an important rule in the synthesis of relatively complex compounds such as, particularly, the (R)-(-) phenylacetylcarbinol (PAC), which is of great economic interest for the manufacture of ephedrine. Here, the R-enantiomere is of interest which is formed by fermentative conversion of pyruvate in the presence of benzaldehyde by means of Saccaromyces erevisiae (DE patent 548439 of 1932).
In this synthesis of PAC by means of yeast cells numerous by-products are formed because of the plurality of enzymes present in the yeast and the cell growth is inhibited by the presence of benzaldehyde.
Also, in this conversion the pyruvate-decarboxylase (PDC) isolated from the yeast yields substantial parts of PAC-isomer 2-hydroxypropiophenons.
The thiamindiphosphate- and Mg.sup.2+ -dependent PDC (E.C. 4.1.1.1) is very common; it is found in many plants, yeasts and fungi and in some bacteria. It is a catalyst for the non-oxidative decarboxylization of pyruvate to acetaldehyde and, in a side reaction, an acyloin condensation occurs wherein .alpha.-hydroxyketones are formed as is apparent from FIG. 1.
Such an enzymatic conversion occurs also on the basis of an aldeyde in place of .alpha.-ketonic carbon acid; the aldehyde formed by the decarbooxylization can also participate as "co-substrate" whereby homo acyloins R-CHOH-CO-R.sup.1 with R=R' is formed.
Also, the PDC was already isolated from Zymomonas mobilis. A comparison of a PDC isolated from yeast with a PDC from Zymamonas mobilis with respect to the formation of PAC under comparable conditions however showed a clearly lower capacity of synthesis of the PDC from Zymomonas mobilis (S. Bringer-Meyer and H. Sahm, Biocatalysis 1 (1988) p. 321-331).
Surprisingly, it has now been determined that, with a particular gene technological modification of the PDC gene from Z. mobilis, a PDC with improved synthesis capacity with respect to the formation of PAC can be obtained which furthermore has a relatively high selectivity for the formation of PAC as compared to 2-hydroxypropiophenone.
The kind of process according to the invention as initially referred to is essentially characterized in that an enzyme is used as a PDC, wherein the tryptophane residue in the substrate canal leading to the active center is replaced by a sterically smaller amino acid residue.
The sterically smaller amino acid residue is particularly a simple, especially an aliphatic amino acid such as, specifically, alanine, glycine, phenylalamine, leucine, isoleucine, arginine or histidine or also serine and threonine.